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. 2019 Apr;100(2):133-138.
doi: 10.1111/iep.12315. Epub 2019 May 6.

Carboxypeptidase A4 promotes proliferation and stem cell characteristics of hepatocellular carcinoma

Hongtao Zhang  1 , Chengfei Hao  1   2 , Haibo Wang  1 , Haitao Shang  1 , Zhonglian Li  1
Affiliations

Carboxypeptidase A4 promotes proliferation and stem cell characteristics of hepatocellular carcinoma

Hongtao Zhang et al. Int J Exp Pathol. 2019 Apr.

Abstract

Carboxypeptidase A4 (CPA4), a member of the metallo-carboxypeptidase family, is overexpressed in liver cancer and is associated with cancer progression. The role of CPA4 in hepatocellular carcinoma (HCC) remains unclear. In this study, we aimed to evaluate the relevance of CPA4 to the proliferation and expression of stem cell characteristics of hepatocellular carcinoma cells. Western blot analysis showed high CPA4 expression in the liver cancer cell line Bel7402 and low expression in HepG2 cells. Knock-down of CPA4 decreased cancer cell proliferation as detected by MTT and clone formation assays. The serum-free culture system revealed that downregulated CPA4 suppressed the sphere formation capacities of tumour cells. However, upregulated CPA4 increased the proliferation and sphere formation capacity. In addition, the protein expression of CD133, ALDH1 and CD44 also increased in cells with upregulated CPA4. In vivo, the overexpression of CPA4 in tumour cells that were subcutaneously injected into nude mice markedly increased the growth of the tumours. These data suggest that CPA4 expression leads to poor prognoses by regulating tumour proliferation and the expression of stem cell characteristics and may therefore serve as a potential therapeutic target of HCC.

Keywords: cancer stem cell; carboxypeptidase A4; hepatocellular carcinoma; proliferation.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Investigating carboxypeptidase A4 (CPA4) expression in liver cancer cell lines and its role in tumour cell proliferation. A, Western blot analysis indicated that CPA4 was highly expressed in the CRC cell line Bel7402 but was lowly expressed in HepG2 cells. CPA4 was successfully downregulated in Bel7402 cells by shRNA and upregulated in HepG2 cells by a CPA4 overexpression plasmid (*P < 0.05). B, The in vitro cell proliferation capacities were detected via MTT and clone formation assays. The MTT assay results showed that the proliferate rate of the Bel7402‐shCPA4 cells (1.60 ± 0.11) was significantly slower than that of the Bel7402 cells (2.20 ± 0.29, P = 0.005), whereas the proliferation rate of HepG2‐CPA4 cells (2.50 ± 0.48) was significantly faster than that of the HepG2 group (1.40 ± 0.49, P = 0.008) (*P < 0.05). C, Clone formation assays indicated that the number and the size of the clones formed by Bel7402‐shCPA4 were lower and smaller than those of Bel7402 cells, and the HepG2‐CPA4 cells formed more and larger clone than the HepG2 cells (*P < 0.05) [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 2
Figure 2
Detection of stem cell properties by sphere formation assays. A, Cell spheres formed after 10 d of culture in ultra‐low adsorption dishes with serum‐free medium. B, The number of round lucent spheres generated from Bel7402 and HepG2‐CPA4 cells was compared with that of Bel7402‐shCPA4 and HepG2 cells (*P < 0.05)
Figure 3
Figure 3
Stem cell marker expression in different cell groups. A, The immunofluorescence staining results showed that Bel7402 cells, which express high levels of carboxypeptidase A4 (CPA4), also showed increased expression of CD133, ALDH1 and CD44. When CPA4 was downregulated in Bel7402 cells, the stem cell marker expression was decreased. B, Upregulated CPA4 in HepG2 cells increased stem cell marker expression [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 4
Figure 4
A mouse xenograft model was utilized to study the effect of carboxypeptidase A4 (CPA4) on tumour growth and how CPA4 correlates with stem cell marker expression in vivo. Mouse tumours were obtained and dissected 4 wk after the subcutaneous injection of the transfected HepG2‐CPA4 and HepG2 cells. A, The comparison of tumour volumes between the HepG2‐CPA4 and HepG2 groups (*P < 0.05). B, The expression of stem cell markers CD133, ALDH1 and CD44 was immunohistochemically evaluated in the HepG2‐CPA4 and HepG2 groups. The expression of CD133, ALDH1 and CD44 was higher in the HepG2‐CPA4 tumour tissues than in the HepG2 tumour tissues [Colour figure can be viewed at wileyonlinelibrary.com]
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